RESUMO
There is a need for simple and reliable method to identify Mycobacterium tuberculosis from AFB smear positive cases. Utility of mycobacterial ES-31 serine protease as a marker to detect Mycobacterium tuberculosis bacilli was explored using Fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. The presence of ES-31 serine protease in bacilli was indicated by green fluorescence on the cell surface. Green fluorescence was observed with M.tb. H37Ra bacilli and M.tb. H37Rv bacilli while no Fluorescence was observed with M. chelonae, Nocardia farcinicum as well as in E. coli showing the usefulness of ES-31 serine protease as a marker for identification of mycobacterium tubercle bacilli in cultures.
RESUMO
PURPOSE: To determine role of antigens released in vivo and in vitro in immunodiagnosis of tuberculosis (TB). METHODS: In vivo released circulating tuberculosis antigen (CTA) was obtained from TB sera by ammonium sulphate precipitation and in vitro released excretory-secretory (ES) antigens from Mycobacterium tuberculosis culture filtrate. CTA and ES antigens were fractionated by SDS-PAGE and electro-eluted gel fractions were analysed for antigen by ELISA. RESULTS: Low molecular weight proteins CTA-9 and ES-9 showed high titre of antigen activity. To explore the diagnostic potential of low molecular weight ES antigen, M. tuberculosis ES antigen was further fractionated by gel filtration chromatography followed by purification on anion exchange column using fast protein liquid chromatography and a highly seroreactive ESG-5D (ES-20) antigen was obtained. Competitive inhibition showed that CTA-9 and ES-9 antigens inhibit the binding of ES-20 antigen to its antibody. Seroanalysis showed sensitivity of 83 and 80% for ES-20 antigen and antibody detection, respectively, in pulmonary TB and 90% in lymph node TB. CONCLUSIONS: Seroreactivity studies using M. tuberculosis ES-20 antigen showed usefulness in detection of TB; in particular, lymph node TB.